Chemical synapses are composed of specialized subcellular structures. Presynaptic terminals contain an organized cytoskeletal architecture to facilitate neurotransmitter release. Most known presynaptic proteins are associated with synaptic vesicles and function in vesicle exocytosis and endocytosis. Few proteins are shown to play roles in organizing presynaptic structure. Work in this laboratory has focused on the molecular events controlling the differentiation of GABAergic motor neurons in the nematode C. elegans. A genetic screen identified several syd genes (for synapse defective) that affect the morphology and organization of the presynaptic terminals in these neurons. Our analysis has suggested that signaling via small GTPases and receptor protein tyrosine phosphatases may function at different steps in the presynaptic structural formation. This proposal focuses on dissecting the signaling pathway involving syd-3, a putative guanine nucleotide exchanger with a Ring-H2 finger. Specifically, we will clone syd-5 and syd-8, both exhibit similar mutant phenotypes as syd-3, suggesting that they may function in the same pathway. We will systematically analyze the ultrastructural defects in selected syd mutants using transmission electron microscopy. This study is the key for understanding how syd genes function in synapse formation. Genes that are involved in the same pathway may be identified as genetic suppressors or enhancers. We have isolated six semi-dominant suppressors of syd-3 and will continue their molecular and genetic characterization. In addition, we will perform a dominant enhancer screen on a temperature sensitive syd-3 mutation, which may identify genes that are sensitive to the dosage of syd-3. GABAergic neurons are essential for brain function. The use of model organisms is invaluable to the studies of the human nervous system. Our results may provide insights into the understanding of synapse formation in all organisms, and may shed light to the search of causes for cancer and other diseases that are associated with synapse malformation.